Cystic Fibrosis (CF) is a prototype for inherited disorders of protein folding. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a polytopic protein expressed in the apical membrane of human epithelial cells. CFTR biogenesis occurs in the endoplasmic reticulum and is facilitated by a complex set of cellular machinery. Greater than 70% of wild type and up to 99% of common mutant forms of CFTR fail to fold properly and are recognized by cellular quality control machinery and degraded by the ubiquitin- proteasome pathway. These observations raise several questions central to CF pathogenesis and treatment. How does cellular machinery coordinate CFTR folding in different cellular compartments? How is misfolded CFTR protein identified by the cell? How is this recognition event coupled to degradation? And how might the efficiency of CFTR folding be improved in patients? The long term goal of this proposal is to characterize the composition, recruitment and function of cellular folding and quality control machinery that regulates the fate of newly synthesized CFTR in the endoplasmic reticulum. The specific aims of this study will use complimentary heterologous expression systems to: l) define the role of the Sec61 translocation machinery in directing early events of CFTR assembly into the ER membrane, 2) identify the dynamic nature of cellular chaperone complexes that govern the fate of newly synthesized CFTR, and 3) examine the role of ER machinery in CFTR degradation by the 26S proteasome complex. Proposed experiments will incorporate photoactive crosslinking probes at precise locations within CFTR to characterize cellular machinery that orients and assembles the nascent chain into the lipid bilayer. Additional studies will use in vitro and in vivo systems to analyze the changing composition of cellular chaperones associated wild type and mutant CFTR during sequential stages of maturation and degradation. Finally lumenal and membrane-bound ER quality control machinery will be identified by biochemical complementation. Together these studies will generate a comprehensive picture of how cellular machinery coordinates and monitors folding events in multiple compartments and ultimately governs the balance between productive and non-productive pathways. Identification of key components that regulate this decision process will be a major step in the development of pharmacologic strategies aimed at improving folding and trafficking of mutant proteins in patients with inherited disorders such as CF.